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In Hot Pursuit

Hogan, Kirk M.D., J.D.

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DENBOROUGH and Lovell1 first reported a myopathy associated with anesthesia nearly 50 yr ago. Despite great strides made in the diagnosis and management of malignant hyperthermia (MH) over the ensuing three decades, the cause itself remained obscure until DNA-based technologies were brought to bear on its many mysteries. To some, the outcome of these investigations may now appear as a surfeit of riches. For example, the number of polymorphisms (i.e., multiple alleles, or DNA sequence variations, of genes within a population) in the gene encoding the calcium release channel (RyR1) of skeletal muscle that are purported to cause MH in at least one human now stands above 100, with more published seemingly by the month.2–4 As acknowledged by the authors of these and similar reports, a causative role in MH for most of the newly detected nucleotide polymorphisms remains to be proven beyond reasonable doubt. But for one at least, i.e., that causing a substitution of a cysteine for arginine at amino acid position 163 (R163C) of the human RyR1 protein, the case is all but closed. In this issue of Anesthesiology, Yang et al.5 use gene targeting to create a mouse line that expresses the human R163C mutation and that exhibits signs of MH at both biochemical and whole animal levels of resolution. Below, the methods underlying this achievement and its broader implications are framed in brief.
To investigate traits correlated with specific nucleotide substitutions, exogenous DNA carrying the polymorphism of interest may be transfected into cultured cells capable of differentiation into an adult mouse. The consequences of the DNA sequence variation may then be compared between manipulated and unmanipulated mice with otherwise identical genetic backgrounds. Gene targeting is the method of introducing a transgene into a desired position of the host genome for site-directed mutagenesis. In embryonic stem (ES) cells, gene targeting creates a mouse in which all of the nucleated cells, including those in the germ line, carry a mutant version of the gene of interest. To generate germ line chimeras, ES cells isolated from a mouse blastocyst are engineered to undergo homologous recombination. This is most often accomplished, as in Yang et al., by electroporation of a cloned region of DNA (i.e., a partial sequence of the RyR1 gene constructed in a targeting vector also carrying genes encoding selectable neomycin and HSV-tk markers) that is closely related or identical to an endogenous region in the genome of the ES cells (in the current case, 129Sv ES cells). Treated ES cells, i.e., the rare 1 in 1,000 cells in which recombination has occurred between the introduced gene and its corresponding chromosomal homolog, are selected in culture from the untreated ES cells, and from those carrying nonhomologous insertions.
The modified and selected ES cells are then injected into the blastocyst of a preimplantation embryo from a different mouse strain (in the current case C57BL/6 blastocysts) and surgically reimplanted into a pseudopregnant foster mother to produce an animal in which the nucleated cells are altered at the desired site. Coat color (i.e., yellow agouti mottling) is used as a marker to determine whether the modified ES cells have contributed to the germ line of the chimera. First-generation offspring are usually heterozygous for the targeted mutation. Backcrossing and interbreeding of chimeras produces mice that may be heterozygous or homozygous for the genetic modification as desired. If mutagenesis results in inactivation of gene expression, the mutation is termed a knock-out. If the altered gene retains its ability to express a functional, albeit modified, protein, the mutation is termed a knock-in. As might be surmised, erisks for failure are inherent at each step of the way, with no a priori guarantee that the transgenic mouse will have a relevant, or even identifiable, phenotype.
In the work of Yang et al., the gamble has paid off. The authors demonstrate that the human R163C RyR1 is transcribed and its protein is expressed in the transgenic mice; that the heterozygous mice become acidotic and febrile and die on exposure to halothane; that dantrolene is fully prophylactic if given before halothane exposure; and that corresponding biochemical changes are observed in myotubes and sarcoplasmic reticulum membranes isolated from the mutant mice. These observations are significant for several reasons. First, the causal property of at least one human RyR1 mutation other than that shared with a spontaneous animal model (i.e., the pig) cannot be doubted. Parallel investigations for all putative human MH mutations, RyR1 or otherwise, are not likely to be forthcoming given the prohibitive time, costs, and risks involved. As the authors have proposed, the creation of transgenic mice with mutations selected from each of the recognized human RyR1 hot spots are well warranted. In turn, a subset of polymorphisms selected for gene targeting that are disproportionately represented in a given human population might also be appended. Expression of the R614C mutation in mice that causes MH as an autosomal dominant trait in humans, and as an autosomal recessive trait in pigs (i.e., R615C), would be of particular interest. Will one or two copies of the mutant gene be necessary and sufficient for expression of MH in the mouse? These and related investigations will be key to detecting differences, if any, between human MH-associated RyR1 mutations expressed in the mouse in their anesthetic drug and dose dependencies, baseline and trigger calcium kinetics, severity of the clinical phenotype, and the like.
Second, as the authors point out, a well-established mouse model has certain advantages over the pig in the planning and conduct of experiments aimed at the contingencies of applied MH research. Among other factors, lower costs, larger sample sizes, and ease of sharing between investigators afforded by murine experiments will facilitate screening of newly introduced inhalational anesthetics, drugs of abuse, and drugs active at the neuromuscular junction for the capacity to trigger MH. As well, a convenient small animal model should speed validation of novel diagnostic tests for the detection of MH susceptibility in humans.
Third, a well-controlled murine model (i.e., human polymorphisms expressed in a highly stable mouse background) may play a crucial role in refining knowledge of the mechanisms of excitation–contraction and their disruption by pharmacologic interventions and genetic variations. Basic MH research is characterized by a large number of “known unknowns.” For example, how do potent anesthetic agents interact with RyR1 and other constituents of the skeletal muscle triad? In multiple species, why does this interaction become lethal in the presence of genetic polymorphisms that otherwise have no measurable influence on the quality of life or on life expectancy? How do divergent mutations in RyR1 present a similar or even identical phenotype? What processes underlie rapid recovery from a catastrophic MH event but leave no residua? Why does the risk for human MH decrease by an order of magnitude with age? Multiple insights into the pathogenesis of MH remain to be disclosed, and it is reasonable to expect that murine models such as that developed by Yang et al. will play a central role.
Kirk Hogan, M.D., J.D.
Department of Anesthesiology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin. khogan@facstaff.wisc.edu
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References

1. Denborough MA, Lovell RRH: Anaesthetic deaths in a family (letter). Lancet 1960; 2:45

2. Ibarra CA, Wu S, Muryama K, Minami N, Ichihara Y, Kikuchi H, Noguchi S, Hayashi YK, Ochiai R, Nishino I: Malignant hyperthermia in Japan: Mutation screening of the entire ryanodine receptor type 1 gene coding region by direct sequencing. Anesthesiology 2006; 104:1146–54

3. Galli L, Orrico A, Lorenzini S, Censini S, Falcianni M, Covacci A, Tegazzin V, Sorrentino V: Frequency and localization of mutations in the 106 exons of the RYR1 gene in 50 individuals with malignant hyperthermia. Hum Mutat 2006;27 8:830–9

4. Robinson R, Carpenter D, Shaw M, Halsall J, Hopkins P: Mutations in RYR1 in malignant hyperthermia and central core disease. Hum Mutat 2006; 27:977–89

5. Yang T, Riehl J, Esteve E, Matthaei KI, Goth S, Allen PD, Pessah IN, Lopez JR: Pharmacologic and functional characterization of malignant hyperthermia in the R163C RyR1 knock-in mouse. Anesthesiology 2006; 105:1164–75

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